1. Field of the Invention
This invention relates to an immunoassay apparatus wherein an antigen-antibody reaction is utilized during the determination of the amount of a specific antigen or a specific antibody contained in a liquid sample, such as blood.
2. Description of the Prior Art
Various immunoassay methods, wherein antigen-antibody reactions are utilized during quantitative determinations of biochemical substances, have heretofore been put into practice. The immunoassay methods can be roughly classified into immunodiffusion methods and labeled immunoassay methods. With the immunodiffusion methods, insoluble complexes formed from antigen-antibody reactions are allowed to sediment in gels and the amounts of the insoluble complexes are determined. A typical one of the immunodiffusion methods is the laser nephelometry utilizing the scattering of a laser beam. With the labeled immunoassay methods, antigens or antibodies are labeled such that their amounts can be determined at sensitivities higher than the sensitivities which can be obtained with the immunodiffusion methods. Radioactive isotopes, bacteriophases, enzymes, fluorescent substances, and metals are employed as the labels.
As one of the labeled immunoassay methods wherein enzymes are employed as the labels, a stepwise reaction method has been proposed. With the stepwise reaction method, a support is used to which an antibody (or an antigen) capable of reacting with a specific antigen (or a specific antibody) has been fixed. A droplet of a liquid sample, which is expected to contain the specific antigen (or antibody), is applied to the support, and the specific antigen (or antibody) is allowed to react with the antibody (or antigen), which has been fixed to the support. Thereafter, a labeled specific antigen (or a labeled specific antibody) is applied to the support. A washing liquid, which contains a substrate capable of being converted into a fluorescent substance by the catalytic action of the label, is then poured on the support. The labeled specific antigen (or antibody), which has not reacted with the antibody (or the antigen), is washed out by the washing liquid. Also, the substrate is converted into the fluorescent substance by the action of the labeled specific antigen (or antibody), which has reacted with the antibody (or the antigen). The amount of the fluorescent substance thus formed is determined. From the amount of the fluorescent substance thus determined, a judgment is made as to the presence or absence or the amount of the specific antigen (or antibody) in the liquid sample.
Also, a sandwich method has been proposed wherein an antigen contained in a liquid sample is allowed to react with an antibody, which has been fixed to a support, and a labeled antibody capable of reacting with the antigen is then applied to the support. After the antigen and the labeled antibody have reacted with each other, the labeled antibody, which has not reacted with the antigen, is washed out by a washing liquid. Additionally, a competitive method has been proposed wherein a labeled antigen or a labeled antibody is added to a liquid sample, and the resulting mixture is then applied to a support.
As one of substance suitable as the substrate, 4-methylumbelliferphosphoric acid (hereinbelow be referred to as 4-MUP) has heretofore been known. Also, as one of substances suitable as the fluorescent substance, 4-methylumbelliferone (hereinbelow be referred to as 4-MU) has heretofore been known.
The labeled immunoassay methods described above are advantageous over the conventional wet processes in that the support markedly facilitates the processing and many liquid samples can be analyzed sequentially and automatically.
In immunoassay apparatuses based on the aforesaid principle, the amount of the fluorescent substance generated in the manner described above is determined from the operations wherein stimulating rays, which have wavelengths falling within a predetermined wavelength range, are irradiated to the support, on which the fluorescent substance has been generated, the fluorescent substance is thereby stimulated by the stimulating rays, and the amount of fluorescence produced by the fluorescent substance is measured photoelectrically.
In the conventional immunoassay apparatuses, in order for the accuracy, with which the amount of the fluorescence is measured, to be improved, the amount of the stimulating rays irradiated to the support is monitored, and a signal representing the amount of the stimulating rays is fed back to the stimulating ray source, which produces the stimulating rays. The amount of the stimulating rays produced by the stimulating ray source is thereby stabilized. Alternatively, the amount of the stimulating rays thus monitored is taken into consideration when a signal representing the amount of the fluorescence is processed. However, with the conventional immunoassay apparatuses, in order for a required level of measurement accuracy to be obtained, the temperature of the whole fluorescence amount measuring section must be controlled such that the fluctuation of the temperature may be within the range of, for example, .+-.1.0.degree. C. Therefore, the conventional immunoassay apparatuses become large in size and complicated.
Also, it has been proposed in CLINICAL CHEMISTRY, Vol. 34, No. 9, pp. 1726-1732, 1988, that light, which is produced by a mercury-vapor lamp and which has a wavelength of .lambda.=365 nm, may be used as the stimulating rays for the stimulation of 4-MU, and the fluorescence produced by 4-MU is passed through an interference filter having a center transmission wavelength of .lambda.=450 nm, or the like, and detected.
However, 4-MU and 4-MUP are simultaneously present on the support, and 4-MUP can also produce the fluorescence. Therefore, when the combination of the stimulating rays having a wavelength of .lambda.=365 nm with the light receiving filter having a center transmission wavelength of .lambda.=450 nm is employed, the problem occurs in that the fluorescence, which is produced by 4-MU and which is to be detected, and the fluorescence, which is produced by 4-MUP and which causes errors to occur in measurement, cannot sufficiently be separated from each other.